Behavioral/Systems/Cognitive SupraspinalGlial ......ponent of the descending nociceptive system that projects to the spinal cord and trigeminal brainstem nuclei and constitutes a major

Behavioral/Systems/Cognitive

Supraspinal Glial–Neuronal Interactions Contribute toDescending Pain Facilitation

Feng Wei, Wei Guo, Shiping Zou, Ke Ren, and Ronald DubnerDepartment of Neural and Pain Sciences, Dental School, Program in Neuroscience, University of Maryland, Baltimore, Maryland 21201

Spinal glial reaction and proinflammatory cytokine induction play an important role in the development of chronic pain states after tissueand nerve injury. The present study investigated the cellular and molecular mechanisms underlying descending facilitation of neuro-pathic pain with an emphasis on supraspinal glial–neuronal relationships. An early and transient reaction of microglia and prolongedreaction of astrocytes were found after chronic constriction injury (CCI) of the rat infraorbital nerve in the rostral ventromedial medulla(RVM), a major component of brainstem descending pain modulatory circuitry. There were prolonged elevations of cytokines tumornecrosis factor-� (TNF-�) and interleukin-1� (IL-1�) after CCI, and they were expressed in RVM astrocytes at 14 d after injury. Intra-RVM injection of microglial and astrocytic inhibitors attenuated mechanical hyperalgesia and allodynia at 3 and 14 d after CCI, respec-tively. Moreover, TNFR1 and IL-1R, receptors for TNF-� and IL-1�, respectively, were expressed primarily in RVM neurons exhibitingimmunoreactivity to the NMDA receptor (NMDAR) subunit NR1. CCI increased TNFR1 and IL-1R levels and NR1 phosphorylation in theRVM. Neutralization of endogenous TNF-� and IL-1� in the RVM significantly reduced CCI-induced behavioral hypersensitivity andattenuated NR1 phosphorylation. Finally, intra-RVM administration of recombinant TNF-� or IL-1� upregulated NR1 phosphorylationand caused a reversible and NMDAR-dependent allodynia in normal rats, further suggesting that TNF-� and IL-1� couple glial hyper-activation with NMDAR function. These studies have addressed a novel contribution of supraspinal astrocytes and associated cytokinesas well as central glial–neuronal interactions to the enhancement of descending facilitation of neuropathic pain.

Key words: astrocyte; TNF-�; IL-1�; NMDA receptor; medulla; nerve injury

IntroductionThe rostral ventromedial medulla (RVM) is an important com-ponent of the descending nociceptive system that projects to thespinal cord and trigeminal brainstem nuclei and constitutes amajor mechanism in the control of pain transmission (Millan,2002; Ren and Dubner, 2002; Fields et al., 2006). Recent studiesindicate that hyperalgesia and allodynia in animal models of per-sistent pain are closely linked to long-lasting activation of de-scending modulatory circuits involving descending facilitation(Porreca et al., 2002; Dubner and Ren, 2004; Gebhart, 2004;Vanegas and Schaible, 2004), which significantly contributes tothe development of persistent pain after peripheral inflammation(Urban and Gebhart, 1999; Wei et al., 1999; Guo et al., 2006) andnerve injury (Pertovaara et al., 1996; Kovelowski et al., 2000).Despite extensive studies on the role of RVM in descending painmodulation, the cellular mechanisms of its involvement in de-scending pain facilitation are poorly understood.

A considerable amount of evidence has demonstrated the ex-istence of dynamic and bidirectional communication betweenglia and neurons at the synapse, suggesting that glia play an activerole in regulation of synaptic transmission in the CNS (Fields and

Stevens-Graham, 2002; Newman, 2003; Perea and Araque, 2006).Glial cells, primarily microglia and astrocytes, exhibit dynamicplasticity by converting from a relatively resting or quiescent stateto a reactive or hyperactive state and appear to modulate neuro-nal activity. Interestingly, after hyperactivation, glia subsequentlyrelease cytokines at the spinal cord (DeLeo et al., 2004; Salter,2004; Sommer and Kress, 2004; Marchand et al., 2005; Watkins etal., 2007) and spinal trigeminal nucleus (Piao et al., 2006; Guo etal., 2007), which may be implicated in central mechanisms ofpersistent pain (Ren and Dubner, 2008). Whether supraspinalglial hyperactivity and their interactions with neurons in theRVM circuitry constitute a mechanism underlying descendingfacilitation during the development of persistent pain has notbeen studied.

Here, we have developed a quantitative behavioral measure ofmechanical hyperalgesia and allodynia associated with a ratmodel of orofacial painful neuropathy, chronic constriction in-jury of the infraorbital nerve (CCI-ION), to investigate the mech-anisms of neuropathic pain with an emphasis on glial–neuronalinteractions in the pain modulatory circuitry. We found a pro-longed astrocytic reaction and increased expression of cytokinestumor necrosis factor-� (TNF-�) and interleukin-1� (IL-1�) inastrocytes and their receptors in RVM neurons after nerve injury.Intra-RVM microinjection of glial inhibitors and neutralizationof endogenous TNF-� and IL-1� significantly attenuated CCI-induced mechanical hyperalgesia and allodynia. We furtherfound that CCI-induced enhancement of NMDA receptor

Received July 30, 2008; accepted Sept. 2, 2008.This work was supported by National Institutes of Health Grants DE18573, 11964, and 15374.Correspondence should be addressed to Dr. Feng Wei, Department of Neural and Pain Sciences, 650 West Balti-

more Street, Room 8259, Baltimore, MD 21201. E-mail: [email protected]:10.1523/JNEUROSCI.3593-08.2008

Copyright © 2008 Society for Neuroscience 0270-6474/08/2810482-14$15.00/0

10482 • The Journal of Neuroscience, October 15, 2008 • 28(42):10482–10495

(NMDAR) subunit NR1 phosphorylation in the RVM was signif-icantly reversed by glial inhibitors or cytokine receptor antago-nists. An elevated NMDAR subunit NR1 phosphorylation in theRVM and moderate behavioral hypersensitivity were also ob-served after microinjection of recombinant TNF-� and IL-1� inthe RVM of normal rats, and were reversed by pretreatment withNMDAR antagonists. This is the first study showing that glial–neuronal interactions coupling proinflammatory cytokines toNMDAR function at the supraspinal level contribute to descend-ing pain facilitation.

Materials and MethodsAnimals. Adult male Sprague Dawley rats weighing 175–350 g (Harlan)were used in all experiments. Rats were on a 12 h light/dark cycle andreceived food and water ad libitum. The experiments were approved bythe Institutional Animal Care and Use Committee of the University ofMaryland Dental School.

Trigeminal neuropathic pain. A model of trigeminal neuropathic painwas made by chronic constriction injury to the unilateral infraorbitalnerve (CCI-ION), which was performed based on the original descrip-tion (Bennett and Xie, 1988) and via an intraoral approach described byImamura et al. (1997). Animals were anesthetized with pentobarbitalsodium (50 mg/kg, i.p.). Surgery was performed under an operationmicroscope. The head of the rat was supine and fixed on a table. A1-cm-long incision was made along the gingivobuccal margin in thebuccal mucosa, beginning immediately next to the first molar. The IONwas freed from surrounding connective tissue by a glass rod and clearlyvisualized using a surgical microscope. At 3– 4 mm from the nerve whereits branches emerge from the infraorbital foramen, the ION was looselytied with two chromic gut (4.0) ligatures, 2 mm apart. This caused minorconstriction of the ION such that the superficial vasculature was mini-mally retarded. The wound was checked for hemostasis and the incisionwas closed using three 4.0 silk sutures. The sham-operated rats receivedonly a unilateral nerve exposure without ligature. All surgical procedureswere performed aseptically. In some cases, a long-lasting anesthetic agent0.25% bupivacaine was injected at the incision sites before and aftersurgery to block local nociceptive inputs induced by acute tissue injuryand then twice per day for another 2 d. Changes in gross behavior andbody weight gain in CCI and sham rats were monitored throughout thestudy and were not significantly different when compared with that innaive rats.

Behavioral testing. All behavioral tests were conducted under blindconditions as previously described (Ren, 1999). The rat was not re-strained but habituated to stand on its hindpaws and lean against theexperimenter’s hand who was wearing a regular leather work glove. Thehabituation required no more than normal petting of the rat, and it wasachieved within one-half an hour. A series of calibrated von Frey fila-ments with bending forces ranging from 9 mg to 118 g were slightlyapplied to the skin within the infraorbital territory, near the center of thevibrissal pad on hairy skin surrounding the mystacial vibrissae. Theseareas were stimulated on both sides: ipsilateral and contralateral to theside where surgery was performed. An active withdrawal of the head fromthe probing filament was defined as a response. Each von Frey filamentwas applied five times at intervals of a few seconds. The response frequen-cies [(number of responses/number of stimuli) � 100%] to a range ofvon Frey filament forces were determined, and a stimulus–response(S–R) curve was plotted. After a nonlinear regression analysis, an EF50

value, defined as the von Frey filament force ( g ) that produces a 50%response frequency, was derived from the S–R curve. We used EF50 valueas a measure of mechanical sensitivity.

Intra-RVM microinjections. As previously described (Guo et al., 2006),animals were anesthetized with 2–3% isoflurane in a gas mixture of 30%O2 balanced with nitrogen, and placed in a Kopf stereotaxic instrument.A midline incision was made after infiltration of lidocaine (2%) into theskin. A midline opening was made in the skull with a dental drill to inserta microinjection needle into the target site. The coordinates for the nu-cleus raphe magnus, the major structure of RVM, were as follows: 10.5mm caudal to the bregma, midline, and 9.0 mm ventral to the surface of

the cerebellum (Paxinos and Watson, 2005). To avoid penetration of thetransverse sinus, the incisor bar was set at 4.7 mm below the horizontalplane passing through the interaural line. Animals were subsequentlymaintained at �1% halothane. Microinjections were performed by de-livering drug solutions slowly over a 10 min period using a 0.5 �l Ham-ilton syringe with a 32 gauge needle. The needle was withdrawn 5 minafter the completion of the injection, and the incision was sutured. Allwound margins were covered with a local anesthetic ointment (Nuper-cainal; Rugby Laboratories). Different groups of animals were subjectedto intra-RVM microinjection with a 0.5 �l volume solution of (1) glialmetabolic inhibitors propentofylline (PPF) (1 fmol, 100 fmol, and 10pmol; Sigma-Aldrich), fluorocitrate (FC) (1 and 100 fmol; Sigma-Aldrich), and minocycline (MC) (10 fmol and 1 pmol; Sigma-Aldrich);(2) cytokine receptor antagonists TNFRI/Fc (TNFRI antagonist; 50 fmol;R&D Systems) and IL-ra (Kineret; IL-1R antagonist; 3 pmol; Amgen); (3)recombinant rat TNF-� (rTNF-�; TNFR agonist; 120 fmol; R&D Sys-tems) or IL-1� (rIL-1�; IL-1R agonist; 120 fmol; PeproTech); and (4)glutamate receptor antagonists MK801 (dizocilpine maleate) (noncom-petitive NMDAR antagonist; 10 pmol; Sigma-Aldrich). All drugs exceptfor fluorocitrate were dissolved or reconstituted in endotoxin-free steriledistilled water, aliquoted, and stored at �70°C. At the time of testing, thestored aliquot was thawed on ice and diluted in sterile 0.9% saline to afinal concentration. The doses of these agents were carefully chosen byreferring to the literature and adjusted by our pilot experiments. Forexample, in the RVM, we found that a dose �0.1 pmol is necessary toavoid an apparent effect of fluorocitrate on neurons (supplemental Fig.S1, available at www.jneurosci.org as supplemental material) (S. Sweitzeret al., 2001; Raghavendra et al., 2003a,b; Ledeboer et al., 2005; Guo et al.,2006, 2007). The control rats underwent identical procedures with injec-tion of the same volume (0.5 �l) of sterile 0.9% saline as vehicle treat-ment. The fluorocitrate solution was prepared as described by Paulsen etal. (1987). The vehicle solution was prepared in the same manner forseparate control experiments, except that fluorocitrate was omitted.

Immunohistochemistry. At different time points after nerve injury, ratswere behaviorally tested to identify CCI-ION-induced mechanical allo-dynia, and then deeply anesthetized with pentobarbital and perfusedtranscardially with 200 ml of saline followed by 500 ml of cold (4°C) 0.1M phosphate buffer containing 4% paraformaldehyde. The brainstemwas removed, immersed in the same fixative overnight at 4°C, and trans-ferred to 30% sucrose (w/v) in phosphate buffer for several days forcryoprotection. Thirty-micrometer-thick coronal sections of the brain-stem were cut with a cryostat at �20°C. Free-floating tissue sectionsincluding RVM were incubated with relevant antibodies overnight. Afterwashes, the sections were incubated with AffiniPure biotinylated second-ary IgG (1:800; Jackson ImmunoResearch Laboratories) for 1 h. Forfluorescence staining, the sections were incubated with streptavidin-Alexa Fluor 568 or 488 (1:600; Invitrogen) for 1 h. For peroxidase iden-tification, the sections were reacted with avidin and biotinylated HRPcomplex (1:200; Vector Laboratories) for 1 h, and reacted with 0.025%diaminobenzidine (Sigma-Aldrich) and 0.003% hydrogen peroxide for5–20 min. Immunostaining control studies were performed by omissionof the primary or secondary antibodies, and by preabsorption with anexcess (10 �g/ml) of the respective antigens. Double labeling was per-formed simultaneously with two primary antibodies obtained from dif-ferent species. After overnight, the sections were incubated for 2 h insolutions containing species-specific secondary antibodies coupled toAlexa 568 or 488, respectively. After washes, all sections were mountedon gelatin-coated slides and coverslipped with Vectashield (Vector Lab-oratories). Images were collected sequentially using a Zeiss fluorescencemicroscope and a charge-coupled device camera controlled by SPOTsoftware. If necessary, a Zeiss 510 MATA laser-scanning confocal micro-scope was further used. Adobe Photoshop (version CS) was used forimage cropping and adjustment.

Western blot. Naive and treated rats were anesthetized with 2% halo-thane and decapitated. The RVM tissues were removed as previouslydescribed (Guo et al., 2006) and homogenized in solubilization buffer(50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100,0.5% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 1 U/ml aprotinin, 2�g/ml leupetin, 2 �g/ml pepstatin A). The homogenate was centrifuged

Wei et al. • Glia in Descending Facilitation of Pain J. Neurosci., October 15, 2008 • 28(42):10482–10495 • 10483

at 20,200 � g for 10 min at 4°C, and the supernatant was removed. Theprotein concentration was determined. Each sample contained proteinsfrom one animal. The proteins (50 �g) were separated on a 7.5% SDS-PAGE gel and blotted to nitrocellulose membrane (GE Healthcare). Theblot was incubated with the respective antibody overnight at 4°C. Themembrane was washed with TBS and incubated for 1 h with anti-goatIgG horseradish peroxidase (HRP) (1:3000; Santa Cruz Biotechnology)in 5% milk/TBS. The immunoreactivity was detected using enhancedchemiluminescence (ECL) (GE Healthcare). The loading and blotting ofequal amount of proteins were verified by reprobing the membrane withanti �-actin antiserum (Sigma-Aldrich). The ECL-exposed films weredigitized, and densitometric quantification of immunoreactive bandswas performed using U-SCAN-IT gel (version 4.3; Silk Scientific).

Antibodies. The following antibodies were used for immunostainingand Western blot: rabbit or mouse anti-glial fibrillary acidic protein(GFAP) (astrocytic marker; 1: 1000; Dako), rabbit anti-S100� (for label-ing astrocytic calcium-binding protein; 1:800; Fitzgerald), mouse anti-OX-42 (for labeling CD11b as microglial marker; 1:800; Serotec), rabbitanti-Iba-1 (for labeling microglial calcium-binding protein; 1:1000;Wako), mouse anti-NeuN (neuronal marker; 1:1000; Millipore Bio-science Research Reagents), goat anti-TNF-� (1:1000; R&D Systems),rabbit anti-IL-1� (1:2000; Millipore Bioscience Research Reagents), goatanti-TNFR1 (1:500; Santa Cruz Biotechnology), rabbit anti-IL-1R (1:500; Santa Cruz Biotechnology), mouse anti-NR1 (1:5000; Millipore),rabbit anti-P-ser896 NR1 (Sigma-Aldrich), and mouse anti-�-actin(Sigma-Aldrich).

Histological reconstruction. The locations of microinjection sites in theRVM were determined by visualization of serial Nissl-stained tissue sec-tions under a microscope. Rats with misplaced microinjection sties wereexcluded from the data analysis or considered as controls in some cases.

Data analysis. Results were expressed as mean � SEM. Statistical com-parisons included Student’s t test or one- or two-way ANOVA with thepost hoc Scheffe F test in Western blot analysis or the Student–Newman–Keuls test in behavioral experiments (ANOVA with repeated measures).In all cases, p � 0.05 was considered to be statistically significant.

ResultsMechanical hyperalgesia and allodynia induced by trigeminalnerve injuryTo probe a role of central glial–neuronal interaction in the devel-opment of persistent pain, we adapted and improved the CCI-ION model in the rat (Vos et al., 1994; Imamura et al., 1997). TheION is a pure sensory nerve, the largest branch of the maxillarydivision of the trigeminal nerve, and innervates the mystacialvibrissae, the hairy vibrissal pad, the upper lip, lateral nose andteeth, and mucosa of the upper jaw (Waite and Tracey, 1995). Toreduce injury related to the surgical procedure and keep the facialskin intact, we performed the CCI-ION operation through anintraoral approach (Imamura et al., 1997). Although the testingof behavioral hyperalgesia and allodynia in spinal models of painis straightforward, assessing nocifensive behavior of the trigemi-nal region is difficult. Moreover, in the CCI-ION model, onlyresponses to noxious thermal stimulation (Imamura et al., 1997)or mechanical stimulation (Kitagawa et al., 2006) have been ex-amined in restrained rats. To reduce the stress of rats in an exper-imental environment, we have developed an appropriate han-dling approach without restraint to assess the mechanicalhyperalgesia and allodynia of the orofacial region in rats (Ren,1999; Sugiyo et al., 2005). The response frequencies to a range ofvon Frey filament forces applied to the ION territory were deter-mined and a S–R curve was plotted (Fig. 1A). An EF50 value wasdefined as the von Frey filament force ( g) that produces a 50%response frequency. Compared with the baseline, the leftwardshift of the S–R curve, resulting in a reduction of EF50, representsmechanical hyperalgesia and allodynia at 1 and 14 d after CCI-ION (Fig. 1A), because there was an increased response to su-

prathreshold stimuli and a decreased response threshold for no-cifensive behavior. Compared with naive and sham-operatedrats, there was a significant reduction of the EF50 values in theipsilateral ION territory from 1 to 28 d after unilateral CCI-IONin rats ( p � 0.001 vs sham group) (Fig. 1B). There were nochanges in the contralateral EF50 values. A moderate and tran-sient (for 1–3 d) reduction of the EF50 values was also seen insham rats ( p � 0.05 vs naive group), and was completely blockedby local anesthesia of the surrounding tissues with 0.25% bupiv-acaine (Moore, 1984) (Fig. 1B), suggesting that the response tothe surgical procedure alone at 1–3 d is caused by input from localtissue after incision and inflammation. Thus, we have shown along-lasting and stable hyperalgesia/allodynia in the CCI-IONrats and used this model to study the involvement of supraspinalglia/cytokines in neuropathic pain of the orofacial origin.

Long-lasting astrocytic hyperactivation in the RVM afternerve injuryTo examine whether nerve injury induced glial reaction in theRVM, we first observed changes in the expression of GFAP, amarker of reactive gliosis and proliferation of astrocytes, in RVMsections from naive rats and after CCI-ION (Fig. 1C). As shownin sample sections at 3 d (Fig. 1Cc) and at 14 d (Fig. 1Cd) afterCCI-ION, increased GFAP immunoreactivity (IR) was seen insmall cell bodies and their processes, compared with naive ani-mals (Fig. 1Ca,b), suggesting prolonged hyperactivation of astro-cytes in RVM after nerve injury. It is known that S100� is a Ca 2�

binding protein that is produced primarily in astrocytes in theCNS. Thus, enhanced S100� expression was also used as a specificfunctional marker of astrocytes in the brain (Tanga et al., 2006).The pattern of enhanced expression of S100� was compared withGFAP expression by immunohistochemistry in the RVM of theCCI-ION (Fig. 1Db– d) and naive rats (Fig. 1Da), beginningfrom 1 to 28 d, further confirming the long-lasting hyperactiva-tion of astrocytes in the RVM after nerve injury. We then usedWestern blot to quantify the time course of changes of GFAPlevels in the RVM tissue (Fig. 1E). Although increased GFAPexpression was found in both sham and CCI rats at 1 and 3 d aftersurgery, the level of GFAP expression was consistently increasedin the RVM at 7 d (by 234.3 � 12.1%; p � 0.001; n � 4 rats), 14 d(by 231.70 � 6.64%; p � 0.001; n � 4), 21 d (by 176.70 � 18.70%;p � 0.05; n � 3), and 28 d (by 194.54 � 8.30%; p � 0.001; n � 3)after CCI but not sham treatment (by 123.10 � 7.38% at 7 d and121.70 � 5.64% at 14 d; p � 0.05; n � 4 per group) comparedwith the naive group (n � 3) (Fig. 1E). Consistent with a smalland short-lasting behavioral hyperalgesia and allodynia at 1 and3 d in the sham-operated rats (Fig. 1B), the transiently increasedGFAP expression in the RVM of the sham-operated rats wasfound at the same time points (Fig. 1E). To distinguish whetherthese early effects in the CCI rats were partially attributable to thetissue incision during the oral surgery, we used local anesthesia ofthe surgical site to block the increased neural activity associatedwith tissue injury during the first 3 d in sham animals. Long-lasting anesthesia with 0.25% bupivacaine totally eliminated theincreased GFAP expression at 3 d after sham operation (n � 3 pergroup) (Fig. 1F), suggesting that the earlier increase in GFAPexpression observed in the RVM at 1 and 3 d after CCI-ION ispartially caused by tissue injury. This observation is also consis-tent with a recent study (Obata et al., 2006) that hindpaw incisionresults in glial hyperactivation in the spinal dorsal horn, begin-ning within 1 d and reaching a maximum hyperactivation at 3 dafter tissue injury.

In a parallel experiment, we extended our analysis to deter-

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mine whether microglial reaction occurs by examining expres-sion of microglial markers in the RVM (Fig. 2). Different fromGFAP expression, CD11b expression in the RVM was primarilyincreased at 1 d (data not shown) and 3 d, but not 14 d and later,

after CCI-ION compared with the naivecondition (Fig. 2A). This early hyperacti-vation of microglia was also confirmed byimmunostaining with a functional micro-glia marker, ionized calcium-bindingadaptor molecule 1 (Iba1) (Fig. 2B). WithWestern blot analysis, an increase inCD11b expression in RVM tissues wasonly detected at early time points (1–3 d)in both CCI and sham-operated rats ( p �0.05; n � 3 per group), compared with na-ive animals (Fig. 2C). Local tissue anesthe-sia also completely blocked CD11b in-crease in the RVM from sham-operatedrats at 3 d (n � 3 per group) (Fig. 2D),suggesting that tissue injury also causedmicroglial hyperactivation. These resultsindicate that nerve and tissue injury acti-vate acutely (1–3 d) both astrocytes andmicroglia in the RVM, both of which arelikely involved in supraspinal mechanismsunderlying the generation of trigeminalneuropathic pain, consistent with previ-ous studies on glial function in the spinalcord (DeLeo and Yezierski, 2001; Raghav-endra et al., 2004; Ledeboer et al., 2006;Obata et al., 2006). In addition, nerve in-jury also induced a prolonged hyperactiva-tion (at least for 28 d) of astrocytes in theRVM. The role of the long-term astrocyticreaction in the RVM during developmentof neuropathic pain was the focus of oursubsequent studies below.

Consistent with previous studies (Piaoet al., 2006; Guo et al., 2007), the timecourse-dependent upregulation of GFAPand CD11b expression was also observedafter CCI-ION in the spinal trigeminal nu-cleus ipsilateral to the nerve injury (datanot shown), a trigeminal site known to re-ceive direct peripheral nociceptive input.Importantly, glial reaction in the RVM isspecifically related to CCI-ION. The facialnucleus lateral to the RVM did not exhibitchanges in GFAP and CD11b expressionafter CCI-ION (data not shown).

Selective inhibition of astrocytes bylower dose of glial toxin fluorocitrateBy its inhibition on aconitase, fluoroci-trate interrupts the tricarboxylic acid(TCA) cycle and can be used as a mito-chondrial inhibitor (Voloboueva et al.,2007). Fluorocitrate-induced glial inhibi-tion results in a variety of metabolic effectsincluding reduced ATP production andincreased glucose metabolism (Fonnum etal., 1997; Hirose et al., 2007). Although notselectively affecting aconitase of different

cellular origin per se, fluorocitrate primarily inhibits TCA cycle ofastrocytes because of more avid uptake by astrocytes and has beenused to assess the importance of glial cells for brain function invivo (Fonnum et al., 1997; Zielke et al., 2007). However, fluoroci-

Figure 1. Mechanical hyperalgesia/allodynia and time course-dependent hyperactivation of astrocytes in the RVM after CCI-ION. A, Examples of S–R function curves illustrating the intensity-dependent head withdrawal responses to mechanical stimuli.There is a significant leftward shift of the curve at 1 d (green) and 14 d (red) after CCI ( p � 0.001; n � 9 rats) compared with thepre-CCI baseline (blue). The leftward shift of the S–R curves indicates an increased response to suprathreshold stimuli and adecrease in EF50 value, suggesting the presence of mechanical hyperalgesia and allodynia. B, Time course of mechanical hyper-algesia and allodynia as indicated by a reduction of the EF50 values. CCI-ION results in mechanical hyperalgesia and allodynia thatpersists over the 28 d observation period. The rats receiving a sham operation show a moderate reduction of the EF50 values thatlasted for 3 d, which is totally blocked by local tissue anesthesia with injection of 0.25% bupivacaine (twice at 1–3 d) at theintraoral incision site in sham-operated rats. CCI-ION versus sham, ***p � 0.001; sham versus naive, #p � 0.05. C, Immunohis-tochemistry of GFAP in the RVM. a, Low power of a tissue section at the rostral medulla level. b, Enlarged RVM region correspond-ing to the small rectangle area in a. Enhanced expression of GFAP is observed at 3 d (c) and 14 d (d) after CCI, compared with GFAPimmunostaining in naive animal (b). D, Immunostaining of S100� in the RVM. Increased S100� expression is observed at 3 d (b),14 d (c), and 28 d (d) after CCI, compared with naive (a). E, Western blots illustrating CCI-induced increase in GFAP in the tissuespunched out from the RVM. A representative blot is shown on top, and the mean protein levels are summarized below. Comparedwith naive and sham-operated (�) rats, the level of GFAP is selectively increased at the 14 and 28 d time points in CCI-ION (�)rats. The increase in GFAP at 1–3 d in sham-operated and CCI-ION rats is consistent with behavioral changes at these time points(B). �-Actin was used as a loading control. The asterisks indicate significant differences from the naive rats (*p � 0.05; n � 3 pertime points). F, Western blots illustrating the effect of bupivacaine, a long-lasting local anesthetic, on the incision-inducedincrease in GFAP expression in the RVM at 3 d after sham operation. Bupivacaine (0.25%; 0.1 ml) is injected at the intraoral incisionsite twice in the first 3 d (�). Saline is injected as a vehicle control (�). Compared with naive rats, the GFAP expression is increasedat 3 d after sham operation (*p � 0.05; n � 3 per group), which is completely abolished by the bupivacaine (n � 3). Scale bars:Ca, 0.5 mm; Cd (for Cb– d), Dd (for Da– d), 0.025 mm. NGC, Nucleus reticularis gigantocellularis; Py, pyramidal tract. Error barsindicate SEM.


trate may also produce neuronal damage(Hornfeldt and Larson, 1990; Largo et al.,1996). Intrastriatal injection of 1 nmol offluorocitrate produces ultrastructural al-terations of astrocytes without affectingneurons, whereas a larger dose at 2 nmolalso affects neuronal structures and me-tabolism (Paulsen et al., 1987).

To search for a safe range of doses forthe selective effect of fluorocitrate on glialcells, we performed double staining for theneuronal marker, NeuN, with terminal de-oxynucleotidyl transferase-mediated bio-tinylated UTP nick end labeling (TUNEL),a label for cell apoptosis, or Fluoro-Jade, amarker of cell necrosis, in RVM sectionsafter microinjection of the drug (supple-mental Fig. S1, available at www.jneurosci.org as supplemental material).When doses of fluorocitrate in the rangebetween 1 fmol (2.2 � 0.41 double-labeledcells/section; n � 4; p � 0.05) and 100 fmol(2.1 � 0.27; n � 4; p � 0.05) (supplemen-tal Fig. S1Ac,C, available at www.jneurosci.org as supplemental material)were used, there was no apparent cellulardamage shown by Nissl (supplemental Fig.S1Aa, available at www.jneurosci.org assupplemental material) and TUNEL stain-ing (supplemental Fig. S1Ab,c, available atwww.jneurosci.org as supplemental mate-rial) except within the track of the micro-injection needle and its nearby area com-pared with that seen in naive (0.25 � 0.16labeled cells/section; n � 4) (supplementalFig. S1C, available at www.jneurosci.org assupplemental material) and vehicle treat-ment (1.38 � 0.18; n � 4) (supplementalFig. S1Ab,C, available at www.jneurosci.org as supplemental material). This finding suggests that a fewdamaged cells may be induced by placement of the injection nee-dle during the microinjection procedure but not by fluorocitrate.However, when a higher dose of fluorocitrate (10 pmol) was in-jected, TUNEL-labeled cells were significantly increased in the RVM(11.88 � 1.26; n � 4; p � 0.001 vs vehicle group; n � 4) (supple-mental Fig. S1Ad,C, available at www.jneurosci.org as supplementalmaterial), with labeled cells in areas far away from the tissue trackof the injection needle. Double immunostaining identified thatall TUNEL-labeled cells were also positive to NeuN (sup-plemental Fig. S1B, available at www.jneurosci.org as supple-mental material). These results suggest that a dose-dependentneuronal apoptosis occurred after microinjection of fluorocitrateinto the RVM. Fluoro-Jade B staining was performed to examinewhether fluorocitrate induces neuronal necrosis in the RVM.Many Fluoro-Jade-positive cells were located in the microinjec-tion sites from rats injected with high (10 pmol; n � 4) but notlow (100 fmol) dose of fluorocitrate (n � 4 per group) (supple-mental Fig. S1D, available at www.jneurosci.org as supplementalmaterial). No double-labeled cells for both Fluoro-Jade B andNeuN were found in the RVM, although Fluoro-Jade-positivecells and NeuN-immunoreactive neurons was also seen in thesame RVM region after microinjection of fluorocitrate (supple-mental Fig. S1D, available at www.jneurosci.org as supplemental

material). This finding suggests that no significant necrosis ofRVM neurons was apparent after microinjection of fluorocitrate.The Fluoro-Jade B-positive population may represent selectivedegeneration of RVM glial cells resulting from toxic effects of ahigh dose of fluorocitrate.

Neuronal damage has been found in the rat spinal dorsal hornafter sciatic nerve injury (Polgar et al., 2005; Scholz et al., 2005).To test whether CCI-ION alone affected the extent of neuronaldamage in the RVM, we also examined TUNEL and Fluoro-Jadestaining in RVM sections from rats with the CCI or sham treat-ment at 7 and 14 d after surgery. In comparison with the naiveanimal, there were no significant differences in the number ofTUNEL- or Fluoro-Jade-labeled cells in the RVM in the CCI- orsham-operated rats ( p � 0.05; n � 4 per group) (data notshown), suggesting that there was no apparent neuronal damagein the RVM produced by the CCI-ION alone.

We then examined the effects of low dose of fluorocitrate onthe glia cells in the RVM of CCI rats (supplemental Fig. S1E,available at www.jneurosci.org as supplemental material). Weconfirmed the lack of any neuronal apoptosis in the RVM sec-tions from 3 d (n � 4 rats) (data not shown) and 14 d (n � 4)(supplemental Fig. S1Ed, available at www.jneurosci.org as sup-plemental material) CCI-treated rats after microinjection of lowdose fluorocitrate (100 fmol) compared with vehicle (n � 3)

Figure 2. Time course-dependent hyperactivation of microglia in the RVM after CCI. A, CD11b immunostaining in the RVM. a,Low power of a section at the rostral medulla level. b, Enlarged RVM region corresponding to the small rectangle area in a. CD11bexpression is enhanced at 3 d (c) but not 14 d (d) after CCI-ION, compared with CD11b immunoreactivity in naive animal (b). B,Iba1 immunostaining in the RVM. Enhanced Iba1 expression is observed at 3 d (b) and declined to near control expression levelsat 14 d (c) and 28 d (d) after CCI, compared with naive animal (a). C, Western blots illustrating CCI-induced short-lasting increasein CD11b in RVM tissues. Compared with naive rats, CD11b expression is temporarily increased at the 1–3 d time points but not at14 and 28 d in both sham-operated (�) and CCI-ION (�) rats. The asterisks indicate significant differences from the naive rats(*p � 0.05; n � 3 per time points). D, Local tissue anesthesia (�), compared with saline injection (�), completely blocks shamoperation-induced increase in CD11b in the RVM at 3 d, when compared with naive rats [sham (�) vs naive, p � 0.05; n � 3 pergroup]. Scale bars: Aa, 0.5 mm; Ad (for Ab– d), Bd (for Ba– d), 0.025 mm. NGC, Nucleus reticularis gigantocellularis. Error barsindicate SEM.


(supplemental Fig. S1Ea, available at www.jneurosci.org as sup-plemental material). With regard to long GFAP turnover time,we examined changes in GFAP protein expression in RVM tissueat 6 h after astrocytic inhibition. Upregulation of GFAP expres-sion in the RVM at 14 d after CCI (n � 4) (supplemental Fig.S1Eb, available at www.jneurosci.org as supplemental material)was decreased after microinjection of fluorocitrate (100 fmol)(n � 4) (supplemental Fig. S1Ee, available at www.jneurosci.orgas supplemental material). In contrast, there was no effect of thisdose of fluorocitrate on the elevated CD11b expression in theRVM sections at 3 d after CCI-treated rats (n � 4) (supplementalFig. S1Ef, available at www.jneurosci.org as supplemental mate-rial) compared with vehicle injection (n � 3) (supplemental Fig.S1Ec, available at www.jneurosci.org as supplemental material).Western blot further confirmed that fluorocitrate (100 fmol) in-

duced the selective inhibition of enhancedGFAP expression [91 � 21.5% (FC; n � 3)vs 238 � 45.9% (vehicle; n � 3); p � 0.05](Fig. 3A) and S100� expression [83.3 �9.5% (FC; n � 3) vs 159.7 � 17% (vehicle;n � 3); p � 0.001] (Fig. 3B) at 14 d afterCCI in the RVM compared with vehicletreatment.

In order to determine whether astro-cytic inhibition by fluorocitrate function-ally reduces cytokine production in theRVM after CCI, we measured changes inTNF-� and IL-1� protein levels in theRVM. Fluorocitrate (100 fmol) totally pre-vented CCI-induced enhancement ofTNF-� expression [86.3 � 27.5% (FC; n �3) vs 241.3 � 30.1% (vehicle; n � 3); p �0.001] (Fig. 3C) and IL-1� expression[109.7 � 16.2% (FC; n � 3) vs 199 �25.4% (vehicle; n � 3); p � 0.01] (Fig. 3D)at 14 d in the RVM compared with vehicletreatment. However, this dose of fluoroci-trate did not affect the basal GFAP (Fig.3A) and S100� (Fig. 3B) and TNF-� andIL-1� protein levels in the RVM tissue at14 d after a sham operation (Fig. 3C,D).

It was shown that intrathecal injectionof fluorocitrate (0.1–1 nmol) reducedinjury-induced elevation of CD11b ex-pression in the spinal cord (Clark et al.,2007; Sun et al., 2008), questioning itspreferential inhibition of astrocytic func-tion. We examined the effect of fluoroci-trate on microglial function in the RVM inour model and with the low dose used tostudy astrocytic function. The resultsshowed that fluorocitrate (100 fmol) didnot block tissue injury-induced enhance-ment of CD11b expression [159 � 5%(FC; n � 3) vs 153 � 3.93% (vehicle; n �3); p � 0.05] (Fig. 3E) at 3 d in the RVMtissues of sham-operated rats, and also didnot prevent nerve injury-induced eleva-tion of CD11b expression [171.3 � 22.1%(FC; n � 3) vs 168.3 � 16.3% (vehicle; n �3); p � 0.05] (Fig. 3E) in the RVM at 3 dafter CCI. In contrast, the enhanced ex-pression of CD11b in the RVM at 3 d after

sham operation or CCI was attenuated or abolished by MC (1pmol) at 4 h after the microinjection (n � 3 per group; p � 0.05)(Fig. 3E) compared with vehicle. Thus, our results suggest thatlow doses of fluorocitrate could be used to locally and effectivelyinhibit hyperactive astrocytes and their function without appar-ent neuronal degeneration and microglial disruption in theRVM.

Time course-dependent effects of glial inhibitors on allodyniaTo identify participation of supraspinal glial hyperactivation inthe cellular mechanisms underlying the initiation and mainte-nance of neuropathic pain, we tested the effects of selective dis-ruption of glial metabolism by microinjection of single doses ofglial inhibitors into the RVM on mechanical hyperalgesia andallodynia at early (3 d) and later time points (14 d) after CCI.

Figure 3. Western blot analysis shows inhibition of fluorocitrate on glial hyperactivation and functions. A, B, Microinjection offluorocitrate (FC�; 100 pmol) significantly inhibits enhanced expression of GFAP (A) and S100� (B) at 14 d after CCI-ION ( p �0.05; n � 3 per group) but does not affect their expression at 14 d after sham operation, compared with vehicle treatment (�).C, D, Fluorocitrate (100 pmol) significantly reverses upregulation of TNF-� (C) and IL-1� (D) at 14 d after CCI-ION ( p � 0.05; n �3 per group) but does not change their expression at 14 d after sham operation, compared with vehicle treatment (�). E, Thereare no effects of FC (100 pmol) on increases in CD11b expression at 3 d after sham operation and CCI-ION, compared with vehicle(Veh) (n � 3 per group). However, the microglial inhibitor MC (1 pmol) attenuates or abolishes CD11b increase in the RVM at 3 dafter sham or CCI treatment. All asterisks indicate p � 0.05 when compared with the naive (n � 3 per group) in A–E. Error barsindicate SEM.


Propentofylline has been known to de-press both microglial and astrocytic acti-vation, reduce proinflammatory cytokinerelease, and attenuate allodynia and hy-peralgesia induced by nerve injury and in-flammation when systemically or intrathe-cally injected (S. M. Sweitzer et al., 2001;Raghavendra et al., 2003a,b; Dorazil-Dudzik et al., 2004; Tawfik et al., 2008).The antiallodynic effect of propentofyllineis associated with a reduction of reactivegliosis involving astroglia and microglia(S. Sweitzer et al., 2001; Raghavendra et al.,2003b; Sweitzer, 2006), which is consistentwith a role of propentofylline as a nonse-lective glial modulator. Minocycline is asemisynthetic tetracycline antibiotic witha number of other properties distinct fromits antimicrobial action including neuro-protection and antiinflammatory action(Elewa et al., 2006). It has been used toevaluate the involvement of microglia inthe development of neuropathic pain, andhas no direct action on astrocytes or neu-rons (Tikka et al., 2001; Raghavendra et al.,2003a; Ledeboer et al., 2005). Recent stud-ies indicate that minocycline reduces mi-croglial migration to cellular debris anddecreases microglial Kv1.3 expression(Nutile-McMenemy et al., 2007).

We injected fluorocitrate, propentofyl-line, and minocycline into the RVM of theCCI and sham-operated rats at 3 and 14 dafter surgery. As shown in Figure 4, allvehicle-treated CCI rats exhibited stableand strong hypersensitivity in behavioraltests (Fig. 4A–C). A single dose of PPF (10pmol) had no effect on EF50 values in thesham animals at 14 d after surgery (Fig.4A, left). However, propentofylline (1fmol, 100 fmol, and 10 pmol) significantlyattenuated mechanical hyperalgesia andallodynia at 14 d after CCI in a dose-dependent manner, lasting for 4 h orlonger (Fig. 4A, left). Similarly, at 3 d afternerve injury, a high dose of propentofyl-line (10 pmol) completely reversed hyper-algesia and allodynia at least for 6 h, andalso transiently blocked moderate behav-ioral hypersensitivity in sham-treated ani-mals at 3 d (Fig. 4A, right). Although it hadno effect on EF50 values in the sham rats at14 d, fluorocitrate (1 and 100 fmol) signif-icantly attenuated mechanical hyperalge-sia and allodynia to an extent similar to thehigh dose of propentofylline at 14 d afterCCI, when compared with the vehicle-treated CCI rats (Fig. 4B, left). Interest-ingly, fluorocitrate at these doses did notreverse hyperalgesia and allodynia ob-served at 3 d after CCI (Fig. 4B, right).Fluorocitrate did produce a trend towardinhibition of behavioral hypersensitivity at

Figure 4. The different effect of glial inhibitors (propentofylline, fluorocitrate, and minocycline) on behavioral hyperalgesia/allodynia at early (3 d) and later (14 d) time points after CCI-ION. At posttreatment, the glial inhibitor propentofylline (A), theselective astrocytic inhibitor fluorocitrate (B), and the microglial inhibitor minocycline (C) are microinjected into the RVM at 3 d(right column) and 14 d (left column) after sham operation or CCI-ION. The dose-dependent effects of the glial inhibitors onbehavioral hyperalgesia/allodynia are measured at 14 d after the surgery. An effective high dose of the inhibitor is then used forbehavioral observation at the 3 d time point. Vehicles are microinjected as a control. A, PPF (1 fmol, 100 fmol, and 10 pmol)produces a dose-dependent attenuation of CCI-induced mechanical hyperalgesia and allodynia at 14 d, persistent for 4 –24 hcompared with vehicle microinjection in CCI rats (left). High-dose propentofylline (10 pmol) has no effect on EF50 values at 14 din sham rats (left). This dose of propentofylline transiently blocks moderate hyperalgesia and allodynia at 3 d compared withvehicle in sham-operated rats (right), and also significantly abolishes mechanical allodynia at 3 d after CCI compared with vehiclein CCI rats (right). B, Two doses of FC (1 and 100 fmol) after microinjection into the RVM significantly attenuate hyperalgesia andallodynia similarly at least for 6 h, compared with vehicle treatment at 14 d in CCI rats (left). High dose of fluorocitrate (100 fmol)has no effect on EF50 values at 14 d in sham rats (left). However, fluorocitrate (100 fmol) does not prevent CCI-induced behavioralhypersensitivity to mechanical stimulation at 3 d after CCI or sham operation (right). C, In contrast, microinjection of MC (1 pmol)into the RVM significantly reduces hyperalgesia and allodynia for 6 h at 3 d after CCI-ION compared with vehicle treatment in CCIrats, and also transiently blocks sham operation-induced mechanical hypersensitivity (right). Lower dose of minocycline (10 fmol)induced a slight and short-lasting inhibition for CCI-induced allodynia (right). The two doses of minocycline have no effect onCCI-induced hyperalgesia and allodynia at 14 d (left). Also, there are no changes in EF50 values after minocycline (1 pmol) injectionat 14 d after sham-operated rats. *** , ###p�0.001; **p�0.01; * , #,ˆ p�0.05 versus CCI plus vehicle in left. ***p�0.001, *p�0.05, CCI plus drug versus CCI plus vehicle in right. #p � 0.05, sham plus drug versus sham plus vehicle in right. Error bars indicateSEM.


3 d in sham animals when compared with the vehicle-treatedsham rats (Fig. 4B, right). In contrast, minocycline (1 pmol)produced inhibition of behavioral hyperalgesia and allodynia,which persisted over the 6 h observation period, at 3 d (Fig. 4C,right) but not 14 d (Fig. 4C, left) after CCI-ION. This dose ofminocycline also transiently and significantly attenuated themoderate hyperalgesia and allodynia induced by sham operationat 3 d (Fig. 4C, right). These results support our hypothesis thatthe development of neuropathic pain behavior in CCI-ION ratsinvolves glial hyperactivation in the RVM. In combination withthe immunohistochemistry and Western blot results, our behav-

ioral analysis further suggests that persis-tent astrocytic hyperactivation in the RVMcontributes to mechanisms underlying themaintenance of neuropathic pain afternerve injury. These results also suggestthat the initiation of behavioral hyperalge-sia and allodynia in the CCI-ION rats pri-marily depends on cellular mechanismsrelevant to microglial reaction in the RVMafter nerve injury and tissue injury. It isknown that minocycline interferes withthe activity of matrix metalloproteinases 2and 9 (MMP-2 and MMP-9) (Machado etal., 2006), which are a family of zinc-dependent proteases responsible for extra-cellular matrix turnover and degradationof bioactive proteins. MMP-2 and MMP-9have been found in dorsal root ganglionneurons and satellite cells and are involvedin induction and maintenance of neuro-pathic pain through IL-1� cleavage andglial hyperactivation at the spinal cord(Kawasaki et al., 2008). Although the dis-tribution of MMPs in the RVM and theirregulation after nerve injury are notknown, we cannot exclude the possible ef-fect of minocycline on glial function viaMMPs and other factors in the RVM.

Enhanced expression of cytokines inRVM astrocytes after nerve injuryOn hyperactivation, astrocytes and micro-glia increase expression and secretion ofproinflammatory cytokines, such asTNF-� and IL-1�. These cytokines havebeen shown to increase synaptic efficacy inthe hippocampus (Woolf and Salter, 2000;Beattie et al., 2002; Viviani et al., 2003;Perea and Araque, 2006, 2007) and con-tribute to neuronal hypersensitivity in spi-nal dorsal horn (DeLeo and Yezierski,2001; Sommer and Kress, 2004; Marchandet al., 2005; Watkins et al., 2007). Hence wenext examined whether astrocytic hyper-activation during the development of neu-ropathic pain is accompanied by an in-crease in cytokine levels in the RVM.Western blots showed that the expressionof TNF-� and IL-1� in the RVM tissueswas significantly upregulated by 58 and62%, respectively, at 14 d after CCI com-pared with sham-operated animals ( p �

0.05; n � 3 per group) (Fig. 5A). An increase of TNF-� and IL-1�immunostaining in the RVM cells was found after CCI-ION (n �6) (Fig. 5B). To verify the source of cells expressing these cytokines,double labeling was performed and showed that both TNF-� IR andIL-1� IR were colocalized with GFAP IR but not NeuN IR in RVMcells at 14 d after CCI-ION (Fig. 5C,D). We did not find coexpressionof the cytokines with CD11b (data not shown), possibly because ofthe very few hyperactivated microglia seen at 14 d. These resultssuggest that hyperactivated astrocytes are a primary source of theincreased TNF-� and IL-1� in the RVM in the maintenance of neu-ropathic pain at 14 d after nerve injury.

Figure 5. Enhanced expression of TNF-� and IL-1� in the RVM at 14 d after CCI-ION. A, Western blots illustrating that the levelsof TNF-� (left) and IL-1� (right) are increased in the RVM in CCI-ION rats compared with sham-operated rats (*p � 0.05; n � 3per group). The anti-TNF-� antibody identifies both the transmembrane TNF-� precursor (26 kDa) and matured TNF-� (17 kDa).Both TNF-� precursor and matured TNF-� exhibit an increase in the RVM at 14 d after CCI-ION. Error bars indicate SEM. B,Immunostaining of TNF-� and IL-1� in RVM. a, d, Low power of a tissue section at the rostral medulla level. b, e, Enlarged RVMregion corresponds to the rectangle area in a and d, respectively. Compared with naive rats (b, e), there is an increase in TNF-� IRand IL-1� IR in RVM at 14 d after CCI-ION (c, f ). The arrowheads indicate positively labeled cells. C, RVM tissue sections at 14 d afterCCI are double stained for TNF-� (a, d; red) and GFAP (b; green) or NeuN (e; green). The arrowheads indicate positively labeledcells. Overlap of a and b reveals that the TNF-� IR is colocalized with GFAP IR in RVM cells (c; yellow, arrows), suggesting itspresence in astrocytes. Overlap of d and e shows a lack of TNF-� in RVM neurons (f ). D, RVM tissue sections were double stainedfor IL-1� (a, d; red) and GFAP (b; green) or NeuN (e; green). The arrowheads indicate positively labeled cells. Overlap of a and breveals that the IL-1�-labeled cells are GFAP immunoreactive in RVM (c; yellow, arrows), suggesting its presence in astrocytes.Overlap of d and e shows a lack of IL-1� IR in these RVM neurons (f ). Scale bars: Ba,d, 0.1 mm; Bb,c,e,f, 0.03 mm; C, D, 0.03 mm.


Increased expression of cytokinereceptors in RVM neurons afternerve injuryAs a possible mechanism of glial–neuronalinteraction, we wanted to know whethercytokine receptors were distributed inRVM neurons as a link between astrocytesand neurons and if these receptors wereupregulated in the RVM after trigeminalnerve injury. Immunostaining showedstronger expression of TNFR1, receptorfor TNF-�, and IL-1RI, receptor for IL-1�,in the RVM at 14 d after CCI (Fig. 6Aa,b,Ca,b, respectively) compared with naiveand sham-operated animals (data notshown). Double labeling indicated thatTNFR1 IR and IL-1RI IR were primarilylocalized in RVM neurons (Fig. 6A,C).Western blot showed that the levels ofTNFR1 and IL-1RI were significantly in-creased at 14 d after CCI when comparedwith naive and sham-operated rats ( p �0.05; n � 3 per group) (Fig. 6B,D). Thesedata demonstrate enhanced expression ofcytokine receptors for TNF-� for IL-1� inthe RVM neurons during maintenance ofneuropathic pain.

Cytokine receptor activation is linked tophosphorylation of NMDARs andmechanical allodynia after nerve injuryWe have shown that glutamate receptorsubunit NMDARs are widely expressedin rat RVM neurons and are upregulated and phosphorylatedin the RVM after tissue injury, contributing to thermal hyper-algesia and mechanical allodynia after peripheral inflamma-tion (Guan et al., 2002; Guo et al., 2006). To provide morpho-logical evidence that supports the interactions between glia/cytokines and neuronal glutamate receptors, we performeddouble immunostaining. The results showed that cytokine re-ceptors TNFR1 and IL-1RI colocalized with NR1 expression, aprincipal component of NMDARs in RVM neurons at 14 dafter CCI-ION (n � 4) (Fig. 7 A, B), similar to the distributionpattern from untreated rats (data not shown). To examinewhether CCI-ION also induces NMDAR activation, the phos-phorylation levels of the NMDAR subunit NR1 were measuredin RVM tissues. As shown in Figure 7C, the immunostainingagainst pNR1ser896 was clearly increased by twofold ( p �0.001; n � 3) at 14 d after CCI-ION compared with the sham-operated (data not shown) and naive rats (n � 3).

To further explore whether astrocytic hyperactivation andconsequent secretion of cytokines contributes to NMDAR acti-vation, we examined the effect of local application of fluorocitrateon increased expression of pNR1 produced by nerve injury.Western blot showed that the increased pNR1ser896 was blockedby fluorocitrate (100 fmol) at 2 h after microinjection into theRVM at 14 d after CCI-ION ( p � 0.01; n � 3) compared withvehicle treatment (n � 3) (Fig. 7D), whereas the same dose offluorocitrate had no effect on pNR1 expression in sham rats (n �3) (Fig. 7D). Moreover, the CCI-induced enhancement of pNR1was also completely eliminated by the soluble anti-TNFR1 IgG, abiological sequester for endogenous TNF-� [TNFR/Fc (T/Fc); 50fmol; n � 3; p � 0.05] (Fig. 7E) and IL-1ra, an IL-1RI antagonist

(3 pmol; p � 0.05; n � 3) (Fig. 7F) at 2 h after intra-RVMinjections, compared with vehicle injections (n � 3). We did notdetect any effects of the immune agents on pNR1 expression inthe RVM tissue in sham-operated rats ( p � 0.05; n � 3 eachgroup) (Fig. 7E,F), when compared with that in naive animals(n � 3). These results suggest that NMDAR activation in theRVM after CCI is facilitated by the activation of RVM astrocytesand the cytokine receptors for TNF-� and IL-1� during themaintenance of neuropathic pain.

To examine whether NMDARs and the cytokine receptorsTNFR1 and IL-1RI contribute to CCI-induced mechanical hy-peralgesia and allodynia, we injected the receptor antagonistsinto the RVM at 14 d after nerve injury. MK801 (10 pmol), anNMDAR channel blocker, did not affect baseline EF50 values insham-treated rats (n � 6) (Fig. 7G); however, it significantlyattenuated mechanical hyperalgesia and allodynia after CCI atleast for 6 h (n � 6; p � 0.05) (Fig. 7G). The posttreatment withTNFR/Fc (50 fmol) or IL-1ra (3 pmol) in the RVM also tempo-rally reversed behavioral hypersensitivity to a similar extent in theCCI rats but did not alter EF50 values recorded in sham rats (Fig.7H). The microinjection of these compounds into the facial nu-cleus near the RVM in CCI-ION rats did not disrupt the devel-opment of behavioral hypersensitivity (data not shown), suggest-ing that the inhibitory effects of the receptor antagonists on CCI-induced hypersensitivity indeed occurred in the RVM but not thesurrounding regions. Thus, we have demonstrated the involve-ment of RVM NMDAR activation in CCI-induced hyperalgesia/allodynia and its dependence on local astrocytic hyperactivationand cytokine release.

Figure 6. Enhanced expression of cytokine receptors for TNF-� and IL-1� in RVM neurons at 14 d after CCI-ION. A, C, Immu-nostaining shows distribution of TNFR1 (A; red) and IL-1RI expression (B; red) in low power of a tissue section at the rostralmedulla level (a) and enlarged RVM region (b) corresponding to the small rectangle area in a. The arrowheads in b and c indicatesingle-labeled neurons. Double labeling shows TNFR1-labeled (Ab) and IL-1RI-labeled cells (Cb) are immunoreactive to NeuN (Ac,Cc; green), suggesting their expression in the RVM neurons (Ad, Cd; arrows). B, D, Western blots illustrating that the levels ofTNFR1 (B) and IL-1RI (D) are increased in the RVM tissues at 14 d in CCI-ION rats compared with sham-operated rats ( p � 0.05;n � 3 per group). *p � 0.05 versus naive in B and D. Scale bars: Aa, Ca, 0.1 mm; Ab– d, Cb– d, 0.03 mm. Error bars indicate SEM.


Intra-RVM exogenous TNF-� and IL-1� produce NMDARactivation and NMDAR-dependent descending facilitationTo test whether the cytokines directly increase NMDAR functionand are implicated in descending pain facilitation, we microin-jected recombinant rat TNF-� (rTNF-�) and IL-1� (rIL-1�) intothe RVM of normal rats and further evaluated their effects onlocal pNR1 expression and mechanical nociception (Fig. 8). Incomparison with vehicle, both exogenous rTNF-� (120 fmol)and rIL-1� (120 fmol) produced a significant upregulation of

NR1 ser896 phosphorylation (by 1.8-foldand 1.71-fold, respectively; p � 0.05; n � 4per group) at 2 h after microinjections(Fig. 8A). The cytokine-induced pNR1 in-crease was completely blocked by pretreat-ment with the NMDAR antagonist MK801(10 pmol; p � 0.05; n � 4 per group) (Fig.8A), suggesting that in addition to activat-ing cytokine receptors at postsynapticsites, these cytokines may enhance gluta-mate release from presynaptic terminalsand glia. Finally, we examined the effectsof exogenous cytokines on mechanical no-ciceptive thresholds (Fig. 8B,C). A singledose of rTNF-� (120 fmol) in the RVMproduced behavioral hyperalgesia and al-lodynia, as indicated by a significant re-duction of EF50 values ( p � 0.05; n � 8)compared with vehicle injection (n � 5)(Fig. 8B). The rTNF-�-produced effect onEF50 began at 30 min and lasted for at least4 h. Microinjection of rIL-1� (120 fmol)produced a similar descending facilitationto a lesser extent (Fig. 8C). The hyperalge-sic/allodynic effects of these cytokineswere completely blocked by pretreatmentwith MK801 (10 pmol; n � 5– 8). MK801alone did not affect EF50 values in normalrats (n � 5) (Fig. 8B). Thus, these resultsfurther confirm that the regulation ofNMDAR activation by locally elevated cy-tokines TNF-� and IL-1� induces hyper-excitability in RVM neurons and contrib-utes to NMDAR-dependent descendingfacilitation of hyperalgesia and allodynia.

DiscussionIn the present study, we observed early mi-croglial hyperactivation at 1–3 d after CCI-ION, followed by a prolonged astrocytichyperactivation in the RVM lasting at leastfor 28 d, with a peak expression at 14 d. Asexpected, inhibition of local glial hyperac-tivation in the RVM using propentofyllinesignificantly attenuated mechanical hy-peralgesia/allodynia at both 3 and 14 d af-ter nerve injury. Interestingly, microinjec-tion of the microglial inhibitorminocycline in the RVM blocked CCI-induced hyperalgesia/allodynia at the earlyphase (3 d) but not the later phase (14 d).In contrast, fluorocitrate, when used atlower doses in the RVM, only attenuatedbehavioral hypersensitivity at the laterphase. Consistent with our finding, studies

with disruption of spinal microglial function by intrathecal injec-tion or local application of minocycline suggest that spinal mi-croglial hyperactivation is required for the initiation, but not themaintenance of nerve injury-induced hyperalgesia (Ledeboer etal., 2005) and evoked neuronal activity in the spinal dorsal horn(Owolabi and Saab, 2006). Similar to CCI-ION-induced glial re-action in the RVM and behavioral hypersensitivity, microglialhyperactivation preceding astrocytic hyperactivation is also ob-

Figure 7. The effect of cytokine inhibitors on NMDAR subunit NR1 phosphorylation in the RVM and behavioral hyperalgesiaand allodynia at 14 d after CCI-ION. A, B, Double immunostaining shows colocalization of TNFR1 (Ab; red) or IL-1RI (Bb; red) withNR1 (Ac, Bc; green) in RVM neurons. Overlay of b and c reveals double labeling of RVM neurons with TNFR1/NR1 (Ad; yellow-orange) or IL-1RI/NR1 (Bd; yellow-orange). Note colocalization of these receptors with NR1 in the cell membrane. C, Comparedwith naive rats, the level of pNR1ser896 is increased at 14 d after CCI-ION ( p � 0.001; n � 3 per group). D, The increasedpNR1ser896 is totally blocked by intra-RVM microinjection of astrocytic inhibitor FC (100 fmol) compared with naive and sham-operated rats (n � 3 per group). E, F, Neutralizing endogenous TNF-� and IL-1� in the RVM using TNFR1/Fc (T/Fc) (50 fmol) (E)and IL-1ra (3 pmol) (F ), respectively, blocks CCI-induced increase in pNR1 expression in the RVM at 14 d after CCI-ION comparedwith sham treatment ( p � 0.05; n � 3 per group). No effect of these inhibitors on basal pNR1 expression is observed insham-operated rats compared with naive rats (n � 3). G, Microinjection of NMDAR channel blocker MK801 (10 pmol) into theRVM abolishes CCI-induced mechanical hyperalgesia and allodynia compared with vehicle (Veh) injection at 14 d after CCI-ION. H,No effect of intra-RVM TNFR1/Fc (50 fmol) and IL-1ra (3 pmol) on basal mechanical threshold is observed in sham-operated ratscompared with naive rats. However, these doses of TNFR1/Fc and IL-1ra attenuate CCI-ION-induced hyperalgesia/allodynia for4 – 6 h after microinjection compared with vehicle treatment in CCI rats. D–F, **p � 0.01; *p � 0.05 versus naive;G, H, *** , ###p � 0.001; ** , ##p � 0.01; * , #p � 0.05 versus CCI plus vehicle. Scale bars: Aa, Ba, 0.015 mm; Ab– d, Bb– d, 0.005mm. Error bars indicate SEM.


served in the spinal cord after inflammation (Raghavendra et al.,2004) and nerve injury (Kawasaki et al., 2008). In addition, recentevidence suggests that the prolonged reaction of astrocytes in thespinal cord plays an important role in maintaining neuropathicpain (Tanga et al., 2006; Zhuang et al., 2006; Kawasaki et al.,2008). Our data further support an important role of RVM mi-croglia in the initiation phase and astrocytes in maintaining me-chanical hyperalgesia/allodynia after nerve injury. Although su-praspinal glial hyperactivation and cytokine expression had beenfound after injury (Raghavendra et al., 2004; Apkarian et al.,2006), no studies have investigated the possibility that they areinvolved in mechanisms underlying descending modulation ofpersistent pain. Thus, we provide the first evidence that a signal-ing sequence from glial hyperactivation, cytokine release, andglutamate receptor phosphorylation in the descending painmodulatory circuitry contributes to the cellular and molecularmechanisms of neuropathic pain.

On glial activation, cytokines including TNF-� and IL-1� aresecreted from glial cells and modulate neuronal activity as chem-ical mediators between glia and neurons. Specifically, TNF-� andIL-1� signaling have been shown to facilitate central glutamatetransmission and potentiate synaptic strength (Beattie et al.,2002; Viviani et al., 2003; Pickering et al., 2005; Yang et al., 2005).The current literature implicates an important role of TNF-� andIL-1� in the peripheral nerve (Zelenka et al., 2005), the spinalcord (Ferreira et al., 1988; Lindenlaub et al., 2000; S. M. Sweitzeret al., 2001; Ohtori et al., 2004), or the trigeminal nucleus (Guo etal., 2007) in the genesis or maintenance of persistent pain de-pending on locally enhanced NMDAR functions (Bursztajn et al.,2004; Guo et al., 2007; Zhang et al., 2008). Our study furthershows that the sustained elevation of TNF-� and IL-1� in theRVM occurred at 14 d after CCI. More importantly, the use ofantagonists for TNF-� receptor and IL-1� receptor neutralize theaction of these endogenous cytokines in the RVM and greatlyattenuate mechanical hyperalgesia and allodynia at 14 d afterCCI. Therefore, these data suggest that secreted TNF-� and IL-1�on astrocytic hyperactivation in the RVM are involved in su-praspinal mechanisms related to the maintenance of neuropathicpain.

NMDAR-containing neurons are widely distributed in theRVM, and NMDAR-dependent descending pain facilitation con-tributes to the development of hyperalgesia after injury (Guo etal., 2006). We have now shown colocalization of cytokine recep-tors TNFR1 and IL-1RI with the NMDAR subunit NR1 in RVMneurons. Neutralizing these cytokines also completely blockedCCI-induced NR1 phosphorylation. Furthermore, recombinantTNF-� and IL-1� induced robust increases in NR1 phosphory-lation in the RVM. Thus, TNF-� and IL-1� associated with glialhyperactivation may affect NMDAR function, similar to othergliotransmitters including chemokines, ATP, and nitric oxide(Pickering et al., 2005; Perea and Araque, 2006, 2007). Couplingof cytokine receptors to the NMDAR may involve several steps.Indeed, recent evidence indicates that intracellular signalingpathways related to Src family tyrosine kinase, PKC (protein ki-nase C), extracellular signal-regulated kinases (ERKs), PSD-95(postsynaptic density-95), phospholipase C, and phospholipaseA2 contribute to IL-1�-induced NMDAR phosphorylation (Vivi-ani et al., 2003, 2006; Guo et al., 2007). Activation of intracellularsignaling pathways involving neuronal pERK and glial p38mitogen-activated protein (MAP) kinases in dorsal root gan-glion, spinal dorsal horn, and the RVM contribute importantly tosynaptic plasticity in central sensitization and the development ofpersistent pain (Zhuang et al., 2005; Katsura et al., 2006; Wei et

Figure 8. Intra-RVM rTNF-� and rIL-1� enhance pNR1 levels and produce descending painfacilitation in normal rats. A, Microinjection of rTNF-� (120 fmol) and rIL-1� (120 fmol) into theRVM enhances expression of pNR1ser896 in the RVM tissue compared with baseline levels innaive rats ( p � 0.05; n � 4 per group). Pretreatment of an NMDAR channel blocker MK801(�) (n � 4) completely prevents rTNF-�- and rIL-1�-induced increase in pNR1 comparedwith vehicle treatment (�). RVM tissues are collected at 2 h after microinjection of the recom-binant agents into the RVM. B, Mechanical sensitivity of the skin is assessed after microinjectionof rTNF-�. The behavioral hyperalgesia and allodynia develops and lasts for 4 h after microin-jection of rTNF-� (120 fmol) as indicated by a reduction of EF50 values ( p � 0.05). Pretreat-ment of MK801 (10 pmol) significantly abolishes rTNF-�-produced mechanical hypersensitivitycompared with vehicle plus rTNF-�. C, Microinjection of rIL-1� (120 fmol) results in a signifi-cant reduction of EF50 values for 2 h ( p � 0.05), which is prevented by pretreatment withMK801. *p � 0.05, rTNF-� or rIL-1� versus vehicle injection. Error bars indicate SEM.


al., 2006; Imbe et al., 2007, 2008; Kawasaki et al., 2008). Wehypothesize that the secreted TNF-� and IL-1� enhance phos-phorylation of NMDARs by binding to their respective receptorsexpressed on NMDAR-containing neurons and trigger the intra-cellular signaling cascades. In addition, both TNF-� and IL-1�also potentiate presynaptic glutamate signaling (Beattie et al.,2002; Viviani et al., 2003; Pickering et al., 2005). Collectively,cytokine TNF-� and IL-1� signaling mediates communicationfrom glial hyperactivation to neuronal hyperexcitability in theRVM by coincidently promoting NMDARs and amplifying glu-tamate signaling. Such supraspinal glial– cytokine–neuronal in-teractions may be critical for the development of descending painfacilitation of neuropathic pain. The role of the MAP kinase sig-naling cascades in RVM glial cells and neurons in the pathogen-esis of neuropathic pain also requires further study.

The present study demonstrated that nerve injury inducedupregulation of TNFR1 and IL-1RI and enhanced phosphoryla-tion of NR1 in RVM, and that microinjection of TNF-� andIL-1� in the RVM of normal rats evoked NMDAR-dependentdescending pain facilitation, suggesting that descending facilita-tion may originate from hyperactivity of RVM neurons that ex-press these cytokine receptors and NMDARs. However, the phys-iological properties of these RVM neurons are unknown. In theRVM, three populations of neurons have been identified basedon the correlation of their action potential firing rates with no-cifensive responses (Fields et al., 1991). On-cells are thought topromote nociception, whereas off-cells to inhibit nociception.Although the role of neutral cells remains unknown, studies sug-gest that they might be involved in pain modulation after inflam-mation (Montagne-Clavel and Oliveras, 1994; Miki et al., 2002).We now know that there are parallel descending facilitatory andinhibitory systems modulating spinal nociceptive transmission(Millan, 2002; Gebhart, 2004; Dubner, 2006). After tissue andnerve injury, not only the enhanced descending facilitation par-allels enhanced descending inhibition from the RVM, but also thenet facilitatory may become dominant, resulting in behavioralhyperalgesia and allodynia (Porreca et al., 2002). Although le-sions of cells expressing �-opioid receptors with dermorphin–saporin conjugate in the RVM do reverse later maintenance ofhyperalgesia (Porreca et al., 2001; Burgess et al., 2002), �-opioidreceptors are found not only on on-cells (Heinricher et al., 1992),but also are located on serotonergic cells (Kalyuzhny et al., 1996)that are considered to be neutral cells in the RVM. Evidence alsoindicates that the majority of spinally projecting serotonergicneurons in the RVM respond to �-receptor agonists (Marinelli etal., 2002; Zhang et al., 2006). At present, it is still difficult toattribute the net effect of descending modulation to a single classof RVM neuronal activity.

It is important to address the finding that fluorocitrate maylead to neuronal damage after application with large doses intocell cultures (Hassel et al., 1995), brain slices (Stone et al., 1990),or in vivo brain tissue (Paulsen et al., 1987; Hornfeldt and Larson,1990; Largo et al., 1996) and even induces animal seizures (Wil-loughby et al., 2003). One reason may be that astrocytic inhibi-tion may lead to accumulation of extracellular glutamate in theCNS (Conti and Weinberg, 1999). We examined possible neuro-nal damage induced by microinjection of fluorocitrate, and ob-served that fluorocitrate caused neuronal apoptosis and glial ne-crosis in the RVM in a dose-dependent manner, which occurredonly when high doses (10 pmol) were used. Consistent with thepresent study, Fonnum et al. (1997) have reported that low dosesof fluorocitrate result in selective and reversible glial disruptionin the striatum, without any ultrastructural evidence of neuronal

damage at synaptic sites. In our experiment, the lower doses offluorocitrate (100 fmol) did not cause cell damage and also hadno effect on function of astrocytes in the RVM of normal animals;however, the lower doses completely abolished upregulation ofGFAP, S100�, TNF-�, and IL-1� levels in the RVM and mechan-ical hypersensitivity at 14 d after CCI. These findings verify thatfluorocitrate inhibits CCI-induced astrocytic hyperactivity at lowdoses after focal application in vivo. Interestingly, long-lastinghyperactivation of astrocytes paralleled an initial upregulation ofglutamate transporter-1 (GLT-1) that is expressed predomi-nantly in astrocytes in the spinal cord at 1–3 d, and downregula-tion of spinal GLT-1 at 7–14 d after nerve injury (Sung et al.,2003; Tawfik et al., 2008; Wang et al., 2008). Propentofyllineprevented the decrease in astrocytic GLT-1 expression in the spi-nal cord at 14 d after nerve injury (Tawfik et al., 2008). Futurestudies are necessary to examine temporal changes in GLT-1 ex-pression in the RVM after nerve injury and whether the glialinhibitors change astrocytic GLT-1 expression after CCI-ION.

ReferencesApkarian AV, Lavarello S, Randolf A, Berra HH, Chialvo DR, Besedovsky

HO, del Rey A (2006) Expression of IL-1� in supraspinal brain regionsin rats with neuropathic pain. Neurosci Lett 407:176 –181.

Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von ZastrowM, Beattie MS, Malenka RC (2002) Control of synaptic strength by glialTNFalpha. Science 295:2282–2285.

Bennett GJ, Xie YK (1988) A peripheral mononeuropathy in rat that pro-duces abnormal pain sensation like those seen in man. Pain 33:87–107.

Burgess SE, Gardell LR, Ossipov MH, Malan TP Jr, Vanderah TW, Lai J,Porreca F (2002) Time-dependent descending facilitation from the ros-tral ventromedial medulla maintains, but does not initiate, neuropathicpain. J Neurosci 22:5129 –5136.

Bursztajn S, Rutkowski MD, Deleo JA (2004) The role of the N-methyl-D-aspartate receptor NR1 subunit in peripheral nerve injury-induced me-chanical allodynia, glial activation and chemokine expression in themouse. Neuroscience 125:269 –275.

Clark AK, Gentry C, Bradbury EJ, McMahon SB, Malcangio M (2007) Roleof spinal microglia in rat models of peripheral nerve injury and inflam-mation. Eur J Pain 11:223–230.

Conti F, Weinberg RJ (1999) Shaping excitation at glutamatergic synapses.Trends Neurosci 22:451– 458.

DeLeo JA, Yezierski RP (2001) The role of neuroinflammation and neuro-immune activation in persistent pain. Pain 90:1– 6.

DeLeo JA, Tanga FY, Tawfik VL (2004) Neuroimmune activation and neu-roinflammation in chronic pain and opioid tolerance/hyperalgesia. Neu-roscientist 10:40 –52.

Dorazil-Dudzik M, Mika J, Schafer MK, Li Y, Obara I, Wordliczek J, Prze-włocka B (2004) The effects of local pentoxifylline and propentofyllinetreatment on formalin-induced pain and tumor necrosis factor-alphamessenger RNA levels in the inflamed tissue of the rat paw. Anesth Analg98:1566 –1573.

Dubner R (2006) Descending modulatory circuitry in the initiation andmaintenance of neuropathic pain. In: Emerging strategies for treatment ofneuropathic pain (Campbell JN, Basbaum AI, Dray A, Dubner R, Dwor-kin RH, Sang CN, eds), pp 123–138. Seattle: IASP.

Dubner R, Ren K (2004) Brainstem mechanisms of persistent pain follow-ing injury. J Orofac Pain 18:299 –305.

Elewa HF, Hilali H, Hess DC, Machado LS, Fagan SC (2006) Minocyclinefor short-term neuroprotection. Pharmacotherapy 26:515–521.

Ferreira SH, Lorenzetti BB, Bristow AF, Poole S (1988) Interleukin-1 as apotent hyperalgesia agent antagonized by a tripeptide analogue. Nature334:698 –700.

Fields HL, Heinricher MM, Mason P (1991) Neurotransmitters in nocicep-tive modulatory circuits. Annu Rev Neurosci 14:219 –245.

Fields HL, Basbaum AI, Heinricher MM (2006) Central nervous systemmechanisms of pain modulation. In: Wall and Melzack’s textbook of pain,Ed 5 (McMahon SB, Koltzenburg M, eds), pp 125–142. New York:Elsevier Churchill Livingstone.

Fields RD, Stevens-Graham B (2002) New insights into neuron-glia com-munication. Science 298:556 –562.


Fonnum F, Johnsen A, Hassel B (1997) Use of fluorocitrate and fluoroac-etate in the study of brain metabolism. Glia 21:106 –113.

Gebhart GF (2004) Descending modulation of pain. Neurosci Biobehav Rev27:729 –737.

Guan Y, Terayama R, Dubner R, Ren K (2002) Plasticity in excitatory aminoacid receptor-mediated descending pain modulation after inflammation.J Pharmacol Exp Ther 300:513–520.

Guo W, Robbins MT, Wei F, Zou S, Dubner R, Ren K (2006) Supraspinalbrain-derived neurotrophic factor signaling: a novel mechanism for painfacilitation. J Neurosci 26:126 –137.

Guo W, Wang H, Watanabe M, Shimizu K, Zou S, LaGraize SC, Wei F,Dubner R, Ren K (2007) Glial-cytokine-neuronal interactions underly-ing the mechanisms of persistent pain. J Neurosci 27:6006 – 6018.

Hassel B, Westergaard N, Schousboe A, Fonnum F (1995) Metabolic differ-ences between primary cultures of astrocytes and neurons from cerebel-lum and cerebral cortex. Effects of fluorocitrate. Neurochem Res20:413– 420.

Heinricher MM, Morgan MM, Fields HL (1992) Direct and indirect actionsof morphine on medullary neurons that modulate nociception. Neuro-science 48:533–543.

Hirose S, Umetani Y, Amitani M, Hosoi R, Momosaki S, Hatazawa J, Gee A,Inoue O (2007) Role of NMDA receptors in the increase of glucose me-tabolism in the rat brain induced by fluorocitrate. Neurosci Lett415:259 –263.

Hornfeldt CS, Larson AA (1990) Seizures induced by fluoroacetic acid andfluorocitrate may involve chelation of divalent cations in the spinal cord.Eur J Pharmacol 179:307–313.

Imamura Y, Kawamoto H, Nakanishi O (1997) Characterization of heat-hyperalgesia in an experimental trigeminal neuropathy in rats. Exp BrainRes 116:97–103.

Imbe H, Okamoto K, Aikawa F, Kimura A, Donishi T, Tamai Y, Iwai-Liao Y,Senba E (2007) Effects of peripheral inflammation on activation of p38mitogen-activated protein kinase in the rostral ventromedial medulla.Brain Res 1134:131–139.

Imbe H, Kimura A, Okamoto K, Donishi T, Aikawa F, Senba E, Tamai Y(2008) Activation of ERK in the rostral ventromedial medulla is involvedin hyperalgesia during peripheral inflammation. Brain Res 1187:103–110.

Kalyuzhny AE, Arvidsson U, Wu W, Wessendorf MW (1996) Mu-opioidand delta-opioid receptors are expressed in brainstem antinociceptivecircuits: studies using immunocytochemistry and retrograde tract-tracing. J Neurosci 16:6490 – 6503.

Katsura H, Obata K, Mizushima T, Sakurai J, Kobayashi K, Yamanaka H, DaiY, Fukuoka T, Sakagami M, Noguchi K (2006) Activation of Src-familykinases in spinal microglia contributes to mechanical hypersensitivityafter nerve injury. J Neurosci 26:8680 – 8690.

Kawasaki Y, Xu ZZ, Wang X, Park JY, Zhuang ZY, Tan PH, Gao YJ, Roy K,Corfas G, Lo EH, Ji RR (2008) Distinct roles of matrix metalloproteasesin the early- and late-phase development of neuropathic pain. Nat Med14:331–336.

Kitagawa J, Takeda M, Suzuki I, Kadoi J, Tsuboi Y, Honda K, Matsumoto S,Nakagawa H, Tanabe A, Iwata K (2006) Mechanisms involved in mod-ulation of trigeminal primary afferent activity in rats with peripheralmononeuropathy. Eur J Neurosci 24:1976 –1986.

Kovelowski CJ, Ossipov MH, Sun H, Lai J, Malan TP, Porreca F (2000)Supraspinal cholecystokinin may drive tonic descending facilitationmechanisms to maintain neuropathic pain in the rat. Pain 87:265–273.

Largo C, Cuevas P, Somjen GG, Martín del Río R, Herreras O (1996) Theeffect of depressing glial function in rat brain in situ on ion homeostasis,synaptic transmission, and neuron survival. J Neurosci 16:1219 –1229.

Ledeboer A, Sloane EM, Milligan ED, Frank MG, Mahony JH, Maier SF,Watkins LR (2005) Minocycline attenuates mechanical allodynia andproinflammatory cytokine expression in rat models of pain facilitation.Pain 115:71– 83.

Ledeboer A, Mahoney JH, Milligan ED, Martin D, Maier SF, Watkins LR(2006) Spinal cord glia and interleukin-1 do not appear to mediate per-sistent allodynia induced by intramuscular acidic saline in rats. J Pain7:757–767.

Lindenlaub T, Teuteberg P, Hartung T, Sommer C (2000) Effects of neutral-izing antibodies to TNF-alpha on pain-related behavior and nerve regen-eration in mice with chronic constriction injury. Brain Res 866:15–22.

Machado LS, Kozak A, Ergul A, Hess DC, Borlongan CV, Fagan SC (2006)

Delayed minocycline inhibits ischemia-activated matrix metalloprotein-ases 2 and 9 after experimental stroke. BMC Neurosci 7:56.

Marchand F, Perretti M, McMahon SB (2005) Role of the immune system inchronic pain. Nat Rev Neurosci 6:521–532.

Marinelli S, Vaughan CW, Schnell SA, Wessendorf MW, Christie MJ (2002)Rostral ventromedial medulla neurons that project to the spinal cordexpress multiple opioid receptor phenotypes. J Neurosci 22:10847–10855.

Miki K, Zhou QQ, Guo W, Guan Y, Terayama R, Dubner R, Ren K (2002)Changes in gene expression and neuronal phenotype in brainstem painmodulatory circuitry after inflammation. J Neurophysiol 87:750 –760.

Millan MJ (2002) Descending control of pain. Prog Neurobiol 66:355– 474.Montagne-Clavel J, Oliveras JL (1994) Are ventromedial medulla neuronal

properties modified by chronic peripheral inflammation? A single-unitstudy in the awake, freely moving polyarthritic rat. Brain Res 657:92–104.

Moore PA (1984) Bupivacaine: a long-lasting local anesthetic for dentistry.Oral Surg Oral Med Oral Pathol 58:369 –374.

Newman EA (2003) New role for astrocytes: regulation of synaptic trans-mission. Trends Neurosci 26:536 –542.

Nutile-McMenemy N, Elfenbein A, Deleo JA (2007) Minocycline decreasesin vitro microglial motility, beta(1)-integrin, and Kv1.3 channel expres-sion. J Neurochem 103:2035–2046.

Obata H, Eisenach JC, Hussain H, Bynum T, Vincler M (2006) Spinal glialactivation contributes to postoperative mechanical hypersensitivity in therat. J Pain 7:816 – 822.

Ohtori S, Takahashi K, Moriya H, Myers RR (2004) TNF-alpha and TNF-alpha receptor type 1 upregulation in glia and neurons after peripheralnerve injury: studies in murine DRG and spinal cord. Spine29:1082–1088.

Owolabi SA, Saab CY (2006) Fractlkine and minocycline alter neuronal ac-tivity in the spinal cord dorsal horn. FEBS Lett 580:4306 – 4310.

Paulsen RE, Contestabile A, Villani L, Fonnum F (1987) An in vivo modelfor studying function of brain tissue temporarily devoid of glial cell me-tabolism: the use of fluorocitrate. J Neurochem 48:1377–1385.

Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates, Ed 5.Amsterdam: Elsevier.

Perea G, Araque A (2006) Synaptic information processing by astrocytes.J Physiol Paris 99:92–97.

Perea G, Araque A (2007) Astrocytes potentiate transmitter release at singlehippocampal synapses. Science 317:1083–1086.

Pertovaara A, Wei H, Hamalainen MM (1996) Lidocaine in the rostroven-tromedial medulla and the periaqueductal gray attenuates allodynia inneuropathic rats. Neurosci Lett 218:127–130.

Piao ZG, Cho IH, Park CK, Hong JP, Choi SY, Lee SJ, Lee S, Park K, Kim JS,Oh SB (2006) Activation of glia and microglial p38 MAPK in medullarydorsal horn contributes to tactile hypersensitivity following trigeminalsensory nerve injury. Pain 121:219 –231.

Pickering M, Cumiskey D, O’Connor JJ (2005) Actions of TNF-� on gluta-mateergic synaptic transmission in the central nervous system. ExpPhysiol 90:663– 670.

Polgar E, Hughes DI, Arham AZ, Todd AJ (2005) Loss of neurons fromlaminas I–III of the spinal dorsal horn is not required for development oftactile allodynia in the spared nerve injury model of neuropathic pain.J Neurosci 25:6658 – 6666.

Porreca F, Burgess SE, Gardell LR, Vanderah TW, Malan TP Jr, Ossipov MH,Lappi DA, Lai J (2001) Inhibition of neuropathic pain by selective abla-tion of brainstem medullary cells expressing the �-opioid receptor. J Neu-rosci 21:5281–5288.

Porreca F, Ossipov MH, Gebhart GF (2002) Chronic pain and medullarydescending facilitation. Trends Neurosci 25:319 –325.

Raghavendra V, Tanga F, DeLeo JA (2003a) Inhibition of microglial activa-tion attenuates the development but not existing hypersensitivity in a ratmodel of neuropathy. J Pharmacol Exp Ther 306:624 – 630.

Raghavendra V, Tanga F, Rutkowski MD, DeLeo JA (2003b) Anti-hyperalgesic and morphine-sparing actions of propentofylline followingperipheral nerve injury in rats: mechanistic implications of spinal glia andproinflammatory cytokines. Pain 104:655– 664.

Raghavendra V, Tanga FY, DeLeo JA (2004) Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflam-matory cytokine expression in the CNS. Eur J Neurosci 20:467– 473.

Ren K (1999) An improved method for assessing mechanical allodynia inthe rat. Physiol Behav 67:711–716.


Ren K, Dubner R (2002) Descending modulation in persistent pain: an up-date. Pain 100:1– 6.

Ren K, Dubner R (2008) Neuron-glia crosstalk gets serious: role in painhypersensitivity. Curr Opin Anesthesiol 21:570 –579.

Salter MW (2004) Cellular neuroplasticity mechanisms mediating pain per-sistence. J Orofac Pain 18:318 –324.

Scholz J, Broom DC, Youn DH, Mills CD, Kohno T, Suter MR, Moore KA,Decosterd I, Coggeshall RE, Woolf CJ (2005) Blocking caspase activityprevents transsynaptic neuronal apoptosis and the loss of inhibition inlamina II of the dorsal horn after peripheral nerve injury. J Neurosci25:7317–7323.

Sommer C, Kress M (2004) Recent findings on how proinflammatory cyto-kines cause pain: peripheral mechanisms in inflammatory and neuro-pathic hyperalgesia. Neurosci Lett 361:184 –187.

Stone EA, Sessler FM, Liu WM (1990) Glial localization of adenylate-cyclase-coupled beta-adrenoceptors in rat forebrain slices. Brain Res530:295–300.

Sugiyo S, Takemura M, Dubner R, Ren K (2005) Trigeminal transitionzone-rostral ventromedial medulla connection and facilitation of orofa-cial hyperalgesia after masseter inflammation in rats. J Comp Neurol495:510 –523.

Sun S, Cao H, Han M, Li TT, Zhao ZQ, Zhang YQ (2008) Evidence forsuppression of electroacupuncture on spinal glial activation and behav-ioral hypersensitivity in a rat model of monoarthritis. Brain Res Bull75:83–93.

Sung B, Lim G, Mao J (2003) Altered expression and uptake activity ofspinal glutamate transporters after nerve injury contribute to the patho-genesis of neuropathic pain in rats. J Neurosci 23:2899 –2910.

Sweitzer S, Martin D, DeLeo JA (2001) Intrathecal interleukin-1 receptorantagonist in combination with soluble tumor necrosis factor receptorexhibits an anti-allodynic action in a rat model of neuropathic pain. Neu-roscience 103:529 –539.

Sweitzer SM, Schubert P, DeLeo JA (2001) Propentofylline, a glial modulat-ing agent, exhibits antiallodynic properties in a rat model of neuropathicpain. J Pharmacol Exp Ther 297:1210 –1217.

Sweitzer SM, Pahl JL, DeLeo JA (2006) Propentofylline attenuatesvincristine-induced peripheral neuropathy in the rat. Neurosci Lett400:258 –261.

Tanga FY, Raghavendra V, Nutile-McMenemy N, Marks A, Deleo JA (2006)Role of astrocytic S100beta in behavioral hypersensitivity in rodent mod-els of neuropathic pain. Neuroscience 140:1003–1010.

Tawfik VL, Regan MR, Haenggeli C, Lacroix-Fralish ML, Nutile-McMenemyN, Perez N, Rothstein JD, DeLeo JA (2008) Propentofylline-induced as-trocyte modulation leads to alterations in glial glutamate promoter acti-vation following spinal nerve transection. Neuroscience 152:1086 –1092.

Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J (2001) Mino-cycline, a tetracycline derivative, is neuroprotective against excitotoxicityby inhibiting activation and proliferation of microglia. J Neurosci21:2580 –2588.

Urban MO, Gebhart GF (1999) Supraspinal contributions to hyperalgesia.Proc Natl Acad Sci U S A 96:7687–7692.

Vanegas H, Schaible HG (2004) Descending control of persistent pain: in-hibitory or facilitatory? Brain Res Rev 46:295–309.

Viviani B, Bartesaghi S, Gardoni F, Vezzani A, Behrens MM, Bartfai T, Bina-glia M, Corsini E, Di Luca M, Galli CL, Marinovich M (2003)Interleukin-1� enhances NMDA receptor-mediated intracellular calciumincrease through activation of the Src family of kinases. J Neurosci23:8692– 8700.

Viviani B, Gardoni F, Bartesaghi S, Corsini E, Facchi A, Galli CL, Di Luca M,Marinovich M (2006) Interleukin-1 beta released by gp120 drives neuraldeath through tyrosine phosphorylation and trafficking of NMDA recep-tors. J Biol Chem 281:30212–30222.

Voloboueva LA, Suh SW, Swanson RA, Giffard RG (2007) Inhibition ofmitochondrial function in astrocytes: implications for neuroprotection.J Neurochem 102:1383–1394.

Vos BP, Strassman AM, Maciewicz RJ (1994) Behavioral evidence of tri-geminal neuropathic pain following chronic constriction injury to therat’s infraorbital nerve. J Neurosci 14:2708 –2723.

Waite PME, Tracey DJ (1995) Trigeminal sensory system. In: The nervoussystem (Paxinos G, ed), pp 705–724. San Diego: Academic.

Wang W, Wang W, Wang Y, Huang J, Wu S, Li YQ (2008) Temporalchanges of astrocyte activation and glutamate transporter-1 expression inthe spinal cord after spinal nerve ligation-induced neuropathic pain. AnatRec (Hoboken) 291:513–518.

Watkins LR, Hutchinson MR, Milligan ED, Maier SF (2007) “Listening”and “talking” to neurons: implications of immune activation for paincontrol and increasing the efficacy of opioids. Brain Res Rev 56:148 –169.

Wei F, Dubner R, Ren K (1999) Nucleus reticularis gigantocellularis andnucleus raphe magnus in the brain stem exert opposite effects on behav-ioral hyperalgesia and spinal Fos protein expression after peripheral in-flammation. Pain 80:127–141.

Wei F, Vadakkan KI, Toyoda H, Wu LJ, Zhao MG, Xu H, Shum FW, Jia YH,Zhuo M (2006) Calcium calmodulin-stimulated adenylyl cyclases con-tribute to activation of extracellular signal-regulated kinase in spinal dor-sal horn neurons in adult rats and mice. J Neurosci 26:851– 861.

Willoughby JO, Mackenzie L, Broberg M, Thoren AE, Medvedev A, Sims NR,Nilsson M (2003) Fluorocitrate-mediated astroglial dysfunction causesseizures. J Neurosci Res 74:160 –166.

Woolf CJ, Salter MW (2000) Neuronal plasticity: increasing the gain inpain. Science 288:1765–1769.

Yang S, Liu ZW, Wen L, Qiao HF, Zhou WX, Zhang YX (2005) Interleukin-1beta enhances NMDA receptor-mediated current but inhibits excitatorysynaptic transmission. Brain Res 1034:172–179.

Zelenka M, Schafers M, Sommer C (2005) Intraneural injection ofinterleukin-1� and tumor necrosis factor-� into rat sciatic nerve at phys-iological doses induces signs of neuropathic pain. Pain 116:257–263.

Zhang L, Sykes KT, Buhler AV, Hammond DL (2006) Electrophysiologicalheterogeneity of spinally projecting serotonergic and nonserotonergicneurons in the rostral ventromedial medulla. J Neurophysiol95:1853–1863.

Zhang RX, Li A, Liu B, Wang L, Ren K, Zhang H, Berman BM, Lao L (2008)IL-1ra alleviates inflammatory hyperalgesia through preventing phos-phorylation of NMDA receptor NR-1 subunit in rats. Pain 135:232–239.

Zhuang ZY, Gerner P, Woolf CJ, Ji RR (2005) ERK is sequentially activatedin neurons, microglia, and astrocytes by spinal nerve ligation and contrib-utes to mechanical allodynia in this neuropathic pain model. Pain114:149 –159.

Zhuang ZY, Wen YR, Zhang DR, Borsello T, Bonny C, Strichartz GR, De-costerd I, Ji RR (2006) A peptide c-Jun N-terminal kinase (JNK) inhib-itor blocks mechanical allodynia after spinal nerve ligation: respectiveroles of JNK activation in primary sensory neurons and spinal astrocytesfor neuropathic pain development and maintenance. J Neurosci26:3551–3560.

Zielke HR, Zielke CL, Baab PJ, Tildon JT (2007) Effect of fluorocitrate oncerebral oxidation of lactate and glucose in freely moving rats. J Neuro-chem 101:9 –16.


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Behavioral/Systems/Cognitive SupraspinalGlial ......ponent of the descending nociceptive system that projects to the spinal cord and trigeminal brainstem nuclei and constitutes a major